On the role of H-NS in the organization of bacterial chromatin: from bulk to single molecules and back.

The chromosomal DNA in the bacterium Escherichia coli is thought to be organized and compacted at least in part as a consequence of the interaction with so-called histone-like or nucleoid-associated proteins. The groups of Stavans and Oppenheim have recently embarked on an ambitious project which aims to quantify the compactive effects of the various members of this group of proteins using magnetic tweezers (Ali et al., 2001). Eventually this could lead to a better understanding of how these proteins work together in the formation of a compact nucleoid. In their most recent study (Amit et al., 2003), they describe the structural effects of H-NS on lambda DNA at the singlemolecule level. Interestingly, their data seem to indicate that H-NS does not induce DNA compaction. Rather, the DNA molecule attains an extended structure upon interaction with H-NS and becomes less flexible. In fact, the effective persistence length is about three times higher than that of naked DNA. The data of Amit et al. (2003) are in striking contrast with recent models about the interaction of H-NS with DNA, which are based both on insights into the structure of the H-NS dimer and microscopic (electron microscopy (EM) and scanning force microscopy (SFM)) observations. H-NS exists as a dimer, which has the ability to selfassociate and form large oligomers (Smyth et al., 2000). The formation of dimers is a result of a leucine zipper-kind of interaction among the N-terminal regions of the two identical monomeric subunits of the protein (Esposito et al., 2002). DNA binding takes place through the C-terminal region (Shindo et al., 1995, 1999). Obviously, within the context of the dimer, two separate DNA binding domains are exposed. It is not exactly clear how H-NS interacts with DNA, but the presence of two DNA binding domains could allow the protein to bind to two DNA strands simultaneously. Large oligomers are thought to be formed by association of dimers in a head-to-tail fashion (Esposito et al., 2002). The DNA binding domains are probably exposed in opposite directions (Esposito et al., 2002), both at the level of a single dimer and at the level of these oligomeric forms of H-NS. Therefore, it is likely that upon initial binding of H-NS oligomers to DNA, only half the number of these domains is used, whereas the others protrude from the opposite side of the H-NS oligomer. A large interaction ‘‘surface’’ is thus still available for binding to another stretch of DNA (within the same or on another DNA molecule—see Fig. 1). Early electron microscopy images suggested coating of DNA by H-NS, but also showed the formation of DNA loops in which distant tracts are apparently brought together by the action of H-NS (Tupper et al., 1994). Subsequently, a number of SFM studies provided further evidence for H-NS as a ‘‘DNA bridge’’ (Dame et al., 2000, 2001, 2002) and showed the functional significance of such bridging (Dame et al., 2001, 2002). A more recent EM study also confirmed these data (Schneider et al., 2001). What does this mean? Should the microscopic data be considered as artifacts, or could there be something particular happening in the magnetic tweezers studies? The most obvious difference between these studies is that the microscopy studies were carried out in bulk, whereas the magnetic tweezers experiments are carried out with one single DNA molecule. As a consequence, the DNA concentration in the single-molecule experiment is extremely low, whereas the H-NS concentrations used for both types of experiments are in the same range (~10 /10 6 M). The fraction of independent binding sites on the DNA molecules occupied by H-NS is determined by the concentrations of protein and DNA and the affinity of the protein for DNA, and follows directly from Le Chatelier’s principle of mass action (Le Chatelier, 1888). The difference between singlemolecule and bulk experiments can be analyzed quantitatively following an approach based on this principle as described in Rippe (1997) or McGhee and von Hippel (1974), depending on the type of binding in the given system (single site, multiple adjacent sites, and cooperative binding). Following Linus Pauling’s adage, ‘‘the student (or the scientist) would be wise to refrain from using the mathematical equation unless he understands the theory that it represents, and can make a statement about the theory that does not consist just in reading the equation. It is fortunate that there is a general qualitative principle, called Le Chatelier’s principle, that relates to all the applications of the principles of chemical equilibrium. When you have obtained a grasp of Le Chatelier’s principle, you will be able to think about any problem of chemical equilibrium that arises, and, by use of a simple argument, to make a qualitative statement about it....’’ (L. Pauling 1964, College Chemistry, 3rd ed., Freeman, San Francisco, CA, 437–438). In following that adage, we limit ourselves to a general and qualitative evaluation of these differences. It follows directly from Le Chatelier’s principle that a different ratio between protein and DNA results in a different degree of saturation of the binding sites on the DNA. In singlemolecule studies such as described here, there is an enormous excess of protein present when compared to bulk Submitted April 10, 2003, and accepted for publication August 7, 2003.